Fate and Concentration Behavior of Aflatoxin M1 During Khoa Manufacture: Development and Validation of a Matrix-Specific HPLC-FLD Method.

Aflatoxin M1 (AFM1), the 4-hydroxylated metabolite of aflatoxin B1, is excreted in the milk of lactating animals fed AFB1-contaminated feed and is of public-health concern because of its well-documented thermal stability during dairy processing. In this study, a matrix-specific extraction, immunoaffinity clean-up, and HPLC-fluorescence detection method were developed and validated for AFM1 in khoa, a traditional heat-desiccated Indian dairy product. The method was linear (R2 ≥ 0.999) over 0.25-20 µg kg- 1, with satisfactory recoveries (83%-102%) and precision (RSD ≤ 6.4 %). Experimentally verified limits of detection and quantification were 0.08 and 0.25 µg kg- 1, respectively. Applying the validated method to AFM1-spiked milk (0.25-20 µg kg- 1) undergoing laboratory-scale khoa manufacture revealed two distinct outcomes: first, a substantial reduction in recoverable AFM1 (63.08%-67.82%) on a total mass basis, likely associated with thermal degradation and/or reduced extractability due to irreversible matrix binding; second, despite this mass loss, moisture evaporation resulted in a net 1.6-1.9-fold increase in AFM1 concentration per kg in the finished khoa. HPLC-FLD and ELISA cross-validation results were not significantly different (paired t-test, p = 0.412; n = 15). These findings establish that thermal processing during khoa manufacture does not completely eliminate AFM1 and that moisture-driven concentration can increase its final concentration per unit weight, highlighting an urgent need for product-specific regulatory limits for heat-concentrated dairy products.